Abnormal salt retention by thick ascending limbs (THALs) causes salt-sensitive hypertension. Flow elevates both stretch and shear stress which stimulates THAL O2- synthesis by NADPH oxidase 4 (NOX4) and NO production by NO synthase 3 (NOS3), respectively. We reported that flow-stimulated THAL Na reabsorption depends on O2- and protein kinase C ? (PKC?) but that flow-induced NO buffers flow-stimulated NaCl reabsorption by directly inhibiting NaCl transport and blunting flow-induced O2-. A high-salt diet increases THAL flow and we reported it enhances THAL NO production by increasing NOS3 expression and activity via de- phosphorylation at threonine 495 (T495), an inhibitory site modified by PKC. Thus salt-induced increases in flow prevent salt-sensitive hypertension via NO. Mechano-transduction of flow-induced stretch and shear stress in epithelia may occur via TRPV4 channels and/or cilia and TRPV4-TRPP2 channels. We showed that flow-induced stretch stimulates TRPV4, increases intracellular Ca (Cai) and stimulates O2-. We also showed that TRPV4 mediates flow-induced increases in Cai and NO. Since cilia and TRPV4-TRPP2 channels sense shear stress, both may be involved in NO production. If so, stretch and shear stress may differentially activate TRPV4 channels and cilia/TRPV4-TRPP2 channels, respectively. This would allow cells to distinguish stretch- and shear stress-elevated Cai. In Dahl salt-sensitive rats (SS) an imbalance between O2- and NO favoring the former causes salt-induced increases in BP but the cause is unknown. Our data show that stretch-induced TRPV4-dependent increases in Cai and flow-induced O2- production are greater in SS than salt-resistant (SR) THALs; however flow-induced NO production is reduced. This decrease is NOT due to scavenging by O2- or differences in NOS3 expression. We also show that a high-salt diet augments the differences in flow-induced Cai and O2- between SS and SR THALs, and causes a difference in NOS3 expression. Thus, we hypothesize that SS THALs display increased TRPV4 channel activity in response to salt-enhanced luminal flow causing abnormally elevated Cai and O2- production by NOX4. Chronically elevated O2- blunts flow-induced NO synthesis due to diminished salt-stimulated NOS3 expression, enhanced NOS3 phosphorylation at T495 and reduced tetrahydrobiopterin (BH4) resulting in salt retention by THALs and salt-sensitivity of BP. We propose 3 aims. Aim 1: Elevating flow and stretch stimulates TRPV4 channel activity more in SS than SR THALs resulting in greater increases in Cai, O2- production by NOX4, PKC? activation and NaCl reabsorption. Aim 2: Chronically elevated O2- in SS THALs blunts the ability of flow-induced shear stress to stimulate NO synthesis as a result of enhanced phosphorylation of NOS3 at T495, reduced BH4, and diminished salt-stimulated NOS3 expression rather than by scavenging or affecting ciliary signaling. Aim 3: Elevated flow-induced O2- and consequent reduced NO production in SS THALs cause salt retention and salt-sensitive hypertension. This proposal may provide a fundamental explanation for disparate data concerning salt-sensitivity of BP in SS.